This invention relates to newly identified mitochondrial polynucleotide sequences having age-dependent mutations, and the use of such polynucleotides, as well as the production and isolation of such polynucleotides. More particularly, methods and compositions useful in modulating and diagnosing age-related disorders are provided.
Mitochondria are organelles primarily responsible for synthesizing ATP (adenosine triphosphate), the chief store of chemical energy for eukaryotic cells. Oxidative phosphorylation is the process by which ATP is synthesized from ADP with the concomitant oxidation of a reduced substrate derived from the breakdown of sugars and fatty acids. The energy for ATP synthesis is provided by the flow of electrons from the reduced substrate along an electron transport system consisting of a series of electron carriers embedded in the inner mitochondrial membrane. The electron transport system catalyzes the transfer of electrons from reduced substrates to molecular oxygen, resulting in substrate oxidation and reduction of oxygen to water.
Mitochondria generate high levels of reactive oxygen species as by-products of the electron transport process and constitute the main source of free radical generation in eukaryotic cells. Free radicals are believed to act as DNA damaging agents and may be responsible for the high mutation rate of mitochondrial DNA (mtDNA). The metabolic importance of mitochondrial genome-encoded protein products is indicative of the significance of mtDNA-associated mutational events. Such events may be pathogenically involved in degenerative processes, Thus, there is a desire to isolate and identify mutated nucleic acid sequences associated with a mitochondrial dysfunction as it relates to the aging process.
The invention relates to the identification and quantification of genetic mutations in a mitochondrial control region that segregate with age-related disorders or are age dependent. The invention provides methods for detecting and quantifying such mutations as a diagnostic for an age related disorder, either before or after the onset of clinical symptoms. More specifically, the present invention provides a method for detecting the presence or risk of age-related disorders by obtaining a biological sample containing mitochondrial nucleic acid from a subject and determining the presence of at least one mutation in a mitochondrial DNA main control region including positions 1 and 660 of the Cambridge sequence (Anderson et al., Nature 250:57, 1981), which correlates with the age of a subject. The invention also involves isolated nucleic acid sequences that are useful in the above-mentioned diagnostics, namely those that correspond, or are complementary, to portions of the mitochondrial control region, and where the sequences contain mutations which correlate with age-related disorders.
The invention provides a method for determining the presence or risk of mitochondrial-associated disease or age dependent mutations in a subject by cleaving nucleic acids in a sample containing mitochondrial nucleic acids with one or more enzymes specific for nuclear nucleic acids in order to eliminate nuclear pseudogenes; amplifying a region of interest by contacting the sample with a pair of primers flanking the region of interest; an subjecting one or more amplified products to an analysis to determine a difference in the amplified product sequence compared to a control sequence, wherein a difference is indicative of a mitochondrial-associated disorder or age dependent mutation. In one embodiment, the method of analysis is by denaturing gradient gel electrophoresis (DGGE). In another embodiment the method of amplification and DGGE is repeated one or more times.
The invention also provides an isolated polynucleotide having a sequence as set forth in SEQ ID NO:1 and having one or more of the following mutations selected from the group consisting of T414G, A368G, a T insertion after position 383, T285C, A249G, T195C, T152C, T146C, variations in length or compositon of the homopolymer tract (HT) D310 at positions 303-315 or variations in length or composition of a CA repeat (positions 514-523), and any combination thereof.
The invention further provides a vector containing a polynucleotide sequence of the invention as well as host cells containing the vector.
The invention also provides a method for detecting an age-related disorder, comprising detecting a mutation in a control region of a mitochondrial polynucleotide sequence, wherein the wild type control region has a sequence as set forth in SEQ ID NO:1.
The invention provides a method for diagnosing a subject having or at risk of having an age-related disorder or mutation by determining the presence of at least one mutation in the nucleic acid sequence of a mitochondrial control region wherein the presence of at least one mutation correlates with risk of having an age-related disorder or mutation.
The invention also provides a method for detecting an age-related mutation of DNA by contacting a sample containing mitochondrial DNA with a nucleic acid probe under conditions that allow the probe and the mitochondrial DNA to hybridize; and detecting hybridization of the probe with the DNA, wherein hybridization of the probe to the DNA is indicative of an age-related mutation.
Also provided a kit useful for the detection of an age-related mutations of mitochondrial DNA comprising carrier means containing therein one or more containers wherein a first container contains a nucleic acid probe that hybridizes to a nucleic acid sequence as set forth in SEQ ID NO:1 wherein SEQ ID NO:1 has one or more of the mutations selected from the group consisting of T414G, A368G, a T insertion after position 383, T285C, A249G, T195C, T152C, T146C, variations in length or compositon of the homopolymer tract (HT) D310 at positions 303-315 or variations in length or composition of a CA repeat (positions 514-523), and combinations thereof
The invention further provides a method for determining the presence or risk of mitochondrial-associated disease or age dependent mutations in a subject by cleaving nucleic acids in a sample containing mitochondrial nucleic acids with one or more enzymes specific for nuclear nucleic acids in order to eliminate nuclear psudogenes; amplifying a region of interest by contacting the sample with a pair of primers flanking the region of interest; and subjecting one or more amplified products to an analysis to determine a difference in the amplified product sequence compared to a control sequence, wherein a difference is indicative of an mitochondrial-associated disorder or age dependent mutation.